The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.

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Renal clearance of quantum dots.

Thermodynamic analysis of the permeability of biological membranes to non-electrolytes

This thesis explores transport phenomena in nanochannels on a chip. Fundamental nanofluidic ionic studies form the basis for novel separation and preconcentration applications for proteomic purposes. The measurements were performed with 50-nm-high 1D nanochannels, which are easily accessible from both sides by two microchannels. Nanometer characteristic apertures were manufactured in the bonded structure of Pyrex-amorphous silicon – Pyrex, in which the thickness of the amorphous silicon layer serves as a spacer to define the height of the nanochannels. The geometry of the nanometer-sized apertures is well defined, which simplifies the modeling of the transport across them. Compared to biological pores, the present nanochannels in Pyrex offer increased stability. Fundamental characteristics of nanometer-sized apertures were obtained by impedance spectroscopy measurements of the nanochannel at different ionic strengths and pH values. A conductance plateau (on a log-log scale) was modeled and measured, establishing due to the dominance of the surface charge density in the nanochannels, which induces an excess of mobile counterions to maintain electroneutrality. The nanochannel conductance can be regulated at low ionic strengths by pH adjustment, and by an external voltage applied on the chip to change the zeta potential. This field-effect allows the regulation of ionic flow which can be exploited for the fabrication of nanofluidic devices. Fluorescence measurements confirm that 50-nm-high nanochannels show an exclusion of co-ions and an enrichment of counterions at low ionic strengths. This permselectivity is related to the increasing thickness of the electrical double layer (EDL) with decreasing salt concentrations, which results in an EDL overlap in an aperture if the height of the nanochannel and the thickness of the EDL are comparable in size. The diffusive transport of charged species and therefore the exclusion-enrichment effect was described with a simple model based on the Poisson-Boltzmann equation. The negatively charged Pyrex surface of the nanometer characteristic apertures can be inversed with chemical surface pretreatments, resulting in an exclusion of cations and an enrichment of anions. When a pressure gradient is applied across the nanochannels, charged molecules are electrostatically rejected at the entrance of the nanometer-sized apertures, which can be used for separation processes. Proteomic applications are presented such as the separation and preconcentration of proteins. The diffusion of Lectin proteins with different isoelectric points and very similar compositions were controlled by regulating the pH value of the buffer. When the proteins are neutral at their pI value, the diffusion coefficient is maximal because the biomolecules does not interact electrostatically with the charged surfaces of the nanochannel. This led to a fast separation of three Lectin proteins across the nanochannel. The pI values measured in this experiment are slightly shifted compared to the values obtained with isoelectric focusing because of reversible adsorption of proteins on the walls which affects the pH value in the nanochannel. An important application in the proteomic field is the preconcentration of biomolecules. By applying an electric field across the nanochannel, anionic and cationic analytes were preconcentrated on the cathodic side of the nanometer-sized aperture whereas on the anodic side depletion of ions was observed. This is due to concentration polarization, a complex of effects related to the formation of ionic concentration gradients in the electrolyte solution adjacent to an ion-selective interface. It was measured that the preconcentration factor increased with the net charge of the molecule, leading to a preconcentration factor of > 600 for rGFP proteins in 9 minutes. Such preconcentrations are important in micro total analysis systems to achieve increased detection signals of analytes contained in dilute solutions. Compared to cylindrical pores, our fabrication process allows the realization of nanochannels on a chip in which the exclusion-enrichment effect and a big flux across the nanometer-sized aperture can be achieved, showing the interest for possible micro total analysis system applications. The described exclusion-enrichment effect as well as concentration polarization play an important role in transport phenomena in nanofluidics. The appendix includes preliminary investigations in DNA molecule separation and fluorescence correlation spectroscopy measurements, which allows investigating the behavior of molecules in the nanochannel itself.

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Transport phenomena in nanofluidics

The microvascular endothelial cell monolayer localized at the critical interface between the blood and vessel wall has the vital functions of regulating tissue fluid balance and supplying the essential nutrients needed for the survival of the organism. The endothelial cell is an exquisite "sensor" that responds to diverse signals generated in the blood, subendothelium, and interacting cells. The endothelial cell is able to dynamically regulate its paracellular and transcellular pathways for transport of plasma proteins, solutes, and liquid. The semipermeable characteristic of the endothelium (which distinguishes it from the epithelium) is crucial for establishing the transendothelial protein gradient (the colloid osmotic gradient) required for tissue fluid homeostasis. Interendothelial junctions comprise a complex array of proteins in series with the extracellular matrix constituents and serve to limit the transport of albumin and other plasma proteins by the paracellular pathway. This pathway is highly regulated by the activation of specific extrinsic and intrinsic signaling pathways. Recent evidence has also highlighted the importance of the heretofore enigmatic transcellular pathway in mediating albumin transport via transcytosis. Caveolae, the vesicular carriers filled with receptor-bound and unbound free solutes, have been shown to shuttle between the vascular and extravascular spaces depositing their contents outside the cell. This review summarizes and analyzes the recent data from genetic, physiological, cellular, and morphological studies that have addressed the signaling mechanisms involved in the regulation of both the paracellular and transcellular transport pathways.

https://www.physiology.org/doi/pdf/10.1152/physrev.00012.2005

Signaling Mechanisms Regulating Endothelial Permeability

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

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The endothelial glycocalyx: composition, functions, and visualization

T HE PENETRATION of capillary walls by water and dissolved substances appears to take place solely by processes which require no energy transformations on the part of the capillary endothelial cells. The rate of net fluid movement across the capillary wall has been shown to be simply proportional to the difference between hydrostatic and osmotic forces acting across the capillary membranes (I, 2). The chemical composition of ascitic fluid (3), edema fluid (4) or glomerular fluid (5) closely resembles that obtainable by filtration of plasma through inert artificial membranes of suitable porosities. Filtration through peripheral capillaries, like that through artificial membranes, varies inversely with the viscosity of the filtrate as this is altered by temperature (6). These striking similarities between the permeability characteristics of living capillaries, on the one hand, and artificial porous membranes on the other, have given rise to the ‘Pore Theory’ of capillary exchange. In its simplest form, the pore theory supposes that the capillary walls are pierced with numerous ultramicroscopic openings which are in general too small to allow the passage of plasma protein molecules, but are of sufficient size and number to account for the observed rates of passage of water and nonprotein constituents of the plasma. Many important questions arise when the pore theory of capillary exchange is examined in detail. The glomerular membranes allow molecules as large as inulin (effective diffusion diameter, d, = 30 A) to pass with no detectable hindrance. Egg albumin (d, = 56 A) passes rapidly through the glomerular membranes (7-9) and even hemoglobin (d, = 62 A) is believed to pass into the glomerular filtrate in appreciable concentration (7, IO). Theoretical and experimental studies on the sieving of molecules by ultrafiltration through porous membranes (I I, 12) indicate that an effective pore diameter of at least IOO A would be required to explain permeability of this magnitude. If this is the case, how are we to imagine the physical structure of the openings in the capillary membranes? Do pores of diameter IOO A penetrate through the endothelial cells and their plasma membranes on both surfaces? Or are the openings confined to the narrow intercellular regions as postulated by Chambers and Zweifach (13)? In the latter case, is the small area available between cells sufficient to explain the observed filtration rates in such rapidly filtering systems as the kidney? If there is a distribution of pore sizes, or if water is free to

E. M. Renkin

Papers

Filtration, diffusion and molecular sieving through peripheral capillary membranes; a contribution to the pore theory of capillary permeability.